Researchers at Rice University have shown that the increasingly popular CRISPR-Cas9 gene-editing tool can be used in human cells in a variety of powerful ways, according to a recent study. The research results have been published in the journal “Nucleic Acids Research”.
Disabled Cas9 (dCas9) proteins have been used by a team led by Rice bioengineer Isaac Hilton and graduate student Kaiyuan Wang to target important regions of the human genome and artificially start transcription of human genes. By using dCas9 to recruit proteins that can naturally transform genes Subsequently, the Rice team was able to reveal important details about human promoters and enhancers, the pieces of our DNA that coordinate when and how well our genes are activated, which in turn controls the behaviors of our cells. “We are using these synthetic biology tools to improve the ability to engineer gene expression and program human cells, and therefore to better understand how our genes work naturally,” Hilton said. “These types of studies are important because, in the long run, this knowledge and technical capability can enable better gene and cell therapies and biotechnologies.”
Hilton said the Nucleic Acids Research study highlights the growing potential of CRISPR-Cas9-based tools for synthetic gene control and cell engineering. The team’s strategy also demonstrates the power of dCas9 to influence and understand the epigenetic factors that drive the human genome. “Only about 2% of our genome contains protein-coding genes, and the remaining 98% is so-called non-coding DNA,” Hilton said. “Enhancers and promoters are key elements of our non-coding genomes and although the vast majority of these elements do not constitute conventional genes, there is fascinating genetic variation in non-coding DNA. This variation gives us the magnificent diversity that allows our species to be both amazing and adaptable.” However, genetic variation in non-coding DNA is also strongly correlated with many diseases, and even subtle differences in these regions can be linked to pathologies,” he said. “A pressing challenge is that it is often very difficult to determine how these differences influence disease onset and treatments.
“Our goal and hope is that technologies and approaches like ours can help researchers get closer to making these important connections, and ultimately predicting and thoughtfully intervening in disease,” Hilton said. By synthetically activating non-coding DNA, the researchers demonstrated how promoters shorten DNA sequences that mark gene start sites and activators can communicate. Remarkably, enhancers can be thousands of base pairs away from their promoters but can stimulate gene transcription by recruiting enhancer proteins and forming direct physical contacts with associated promoters. “Enhancers can also sometimes create mysterious transcripts called activator RNAs (eRNAs),” Hilton said. “Kai has shown that CRISPR technologies can be used to activate these eRNAs, and that in some cases this promotes a type of genomic tracking, in which an activator can be dragged along DNA to engage with promoters downstream.” It also appears that along the way, vital transcriptional and epigenetic information can be deposited,” he said. “It is exciting to speculate that this information could serve as a kind of gene expression bookmark that reinforces subsequent transcription cycles in a type of positive epigenetic feedback.
Their strategy revealed that enhancers and promoters can have “intrinsic reciprocity.” While they knew that signals can be passed from an enhancer to a promoter, they learned that this transmission can also go the other way. “We see regulation happening from a promoter to an upstream enhancer,” Wang said. “Mechanically, this is considered non-canonical and therefore rather surprising.” They also found that CRISPR enhancers can increase the frequency of physical contact between enhancers and promoters, but only when targeted to an enhancer, suggesting a kind of one-way street to increase physical contact. “We now know that these pieces of DNA can send messages back and forth, but there appears to be a significant aspect of directionality for physical contact,” Hilton said. “There is certainly reciprocity, but it seems that the predominant mode of regulation here is one in which an enhancer directs to one or more corresponding promoters.” The researchers said their study was only possible because of advances in CRISPR-Cas9. “Without these genomic targeting tools, we would have had to use other more invasive and disruptive synthetic methods, such as eliminating or genetically modifying a regulatory element,” Wang said. “Our approaches here facilitate epigenetic hijacking or reusing native cellular mechanisms to understand and engineer precisely how genes are controlled.” (ANI)
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